Banana bunchy top disease (BBTD) is the most important virus disease of bananas (Dale, 1987, Advances in Virus Research 33 301-325), The disease is widespread in Asia and the South Pacific and has limited distribution in Australia and Africa. It has not been reported from the Americas. The disease was originally assumed to be caused by a luteovirus as it was persistently aphid transmitted but not mechanically transmitted, induced yellows type symptoms and infected plants that had damaged phloem. However, recently 18-20 nm isometric virus-like particles (VLPs) have been purified from infected plants and have been demonstrated to be associated with the disease (Harding et al., 1991, Journal of General Virology 72 225-230; Thomas & Dietzgen, 1991, Journal of General Virology 72 217-224; Wu and Su, 1990, Journal of Phytopathology 128 153-160). Harding et al (Harding et al., 1991, Journal of General Virology 72 225-230; Harding et al., 1993, Journal of General Virology 74 323-328) have isolated circular, single-stranded DNA of about 1 kb from these VLPs and cloned and sequenced one ssDNA component. This component, known as BBTV DNA component 1, had one large open reading frame (ORF) in the virion sense and encoded a putative replicase. This component was transmitted with the disease via aphids.
In Burns et al., 1994, Arch Virol. 137 371-380, they report the cloning and sequencing of a second component of BBTV. They found that a 93 nucleotide sequence was strongly conserved between the two ssDNA genomic components of BBTV. Two outwardly extending degenerate primers were designed from this sequence and used in a polymerase chain reaction (PCR) with DNA extracted from purified BBTV virions. PCR amplified products consisting of at least seven distinct bands all approximately 1 kb and possibly representing full-length BBTV dsDNA were resolved. The PCR amplified products were cloned and the clones screened by restriction enzyme analysis. Four distinct restriction analysis groups were identified. This reference concluded that the genome of BBTV contains at least five components and that BBTV belongs to a previously undescribed group of plant viruses which may also contain subterranean clover stunt virus.
In Karan et al., 1994, Journal of General Virology 75 3541-3546, mention is made of BBTV component 1 from isolates from 10 different countries being cloned and sequenced and the sequences were subsequently aligned and compared. This analysis indicated two groups: the South Pacific group (isolates from Australia, Burundi, Egypt, Fiji India, Tonga and Western Samoa) and the Asian group (isolates from the Philippines, Taiwan and Vietnam). The mean sequence difference within each group was 1.9 to 3.0% and between isolates from the two groups were approximately 10%, but some parts of the sequences differed more than others. However, the protein encoded by the major open reading frame differed by approximately 5%. The region from the beginning of the stem-loop sequence to the potential TATA box was identical in all isolates except for a two nucleotide change in the Western Samoan isolate and a single change in that of the NSW isolate. These results, together with other evidence, suggest that BBTV has spread to bananas after the initial movement of bananas from the Asian Pacific regions to Africa and the Americas.
In Xie et al., 1995, Phytopathology 85 339-347, the Hawaiian isolate of BBTV was purified from infected banana cultivar Williams. Three single-stranded DNA (ssDNA) components were cloned and sequenced; they were named component 1, 3 and 4 respectively. Component 1 is 1,110 nucleotides in length and shares 98% nucleotide sequence identity with the BBTV DNA component 1 of the Australian isolate as described In Harding et al. (1993) above. This component contains two open reading frames (ORF) capable of encoding a protein of 33.5 kDa, which may function as a replicase, and a protein about 15.2 kDa, with unknown functions. Component 3 is 1,057 nucleotides in length and does not contain any ORFs larger than 10 kDa. Component 4 is 1,017 nucleotides in length and potentially encodes a protein of 18.9 kDa. All three ssDNA components have a same stem-loop sequence and have a conserved non-coding region. The sequence of each of these-three components is different from that of BBTV DNA components of two Taiwanese isolates. BETV-specific clones were used in dot-blot hybridisation assays for detection of BBTV in plants using radioactive and non-radioactive probes. A polymerase chain reaction (PCR) assay was developed for detection of BBTV in banana samples and single aphids. Dot-blot hybridisation assays were as sensitive as enzyme-linked immunosorbent assay (ELISA) while PCR was 1,000 times more sensitive than dot-blot and ELISA assays for detection of BBTV in bananas.
Although so called intergenic regions of genomic components 1, 3 and 4 have been detected in Harding et al., 1993 supra; and Xie and Hu, 1995, supra, the only charcteristic of such intergenic regions has been a stem-loop structure in component 1 as disclosed in Harding et al., 1993, supra.
In an earlier application, (i.e. International Application PCT/AU95/00311) reference is made to BBTV components 3, 4 and 6 which are claimed per se. Regions outside the ORF regions (i.e. the so called "intergenic regions") have also been disclosed in this earlier application. Genomic components 1, 3 and 4 disclosed in Xie and Hu, 1995, supra correspond to components 1, 2 and 5 referred to in the earlier application PCT/AU95/00311.